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1.
建立一种以EV71 3C蛋白酶为靶标的抗肠病毒药物筛选模型,并应用于小分子化合物库筛选具有抗EV71活性的化合物.从临床手足口病例标本中分离肠道病毒进行PCR鉴定及基因组测序.通过插入突变在黄色荧光YFP编码框合适位点处引入EV71 3C酶切位点,构建对3C蛋白酶敏感的报告质粒pc DNA3-m YFP,然后将其与表达3C的质粒共转293A细胞,在3C抑制剂Rupintrivir存在与否的情况下通过荧光显微镜和酶标仪检测Ex(500nm)/Em(535nm)荧光信号的变化,判断建模是否成功;利用建好的筛选模型在高通量药物筛选平台对小分子化合物库进行初筛和复筛;再利用空斑分析检测筛选出的活性化合物是否对临床分离的EV71毒株具有抑制作用.m YFP在293A细胞中表达良好,3C的表达使荧光信号下降80%,Rupintrivir的存在则几乎不影响荧光表达,说明以3C为靶位的筛选模型构建成功.经过高通量初筛和复筛从26 000多种小分子化合物中获得26种能够显著回复m YFP表达的活性化合物;空斑分析显示其中2种化合物具有较为明显的抑制EV71复制的活性.因此,我们所构建的3C-m YFP共表达系统是一种简便有效的、可用于高通量筛选抗EV71 3C~(pro)药物的筛选模型. 相似文献
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Huajun Li Shuxian Li Jianfeng Zheng Chunyan Cai Bin Ye Jun Yang Zhimin Chen 《Microbiology and immunology》2015,59(3):152-159
Enterovirus 71 (EV71) infection can cause severe neurological complications including meningoencephalitis (ME) in some patients with hand, foot and mouth disease (HFMD). However, to date no studies have reported changes in cytokine concentrations and their correlations with clinical variables in patients with ME following EV71 infection. In this study, responses of Th1/Th2 cytokine, including IL‐2, IL‐4, IL‐6, IL‐10, TNF‐α and IFN‐γ, in cerebrospinal fluid (CSF) from patients with EV71‐related HFMD with ME and patients with febrile convulsions (FC) were analyzed using cytometric bead array technology. It was found that CSF IL‐6 and IFN‐γ concentrations were significantly higher in patients with EV71‐related ME than in those with FC. Additionally, both CSF IL‐6 and IFN‐γ concentrations were correlated with CSF cytology, fever duration and duration of hospital stay. More interestingly, a positive correlation between CSF IL‐6 and IFN‐γ concentrations was observed. Finally, receiver operating characteristic analysis revealed that when a cutoff value of 9.40 pg/mL was set for IL‐6, the sensitivity and specificity were 84.5% and 85.5%, respectively, for discriminating EV71‐related ME from FC. In conclusion, IL‐6 and IFN‐γ may be associated with EV71‐induced neuropathology. 相似文献
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Zhihui Li Bin Cui Xiaowen Liu Laicheng Wang Qingjie Xian Zhaoxi Lu Shuntao Liu Yinguang Cao Yueran Zhao 《Microbiology and immunology》2020,64(3):189-201
Enterovirus 71 (EV71) is the predominant pathogen for severe hand, foot, and mouth disease (HFMD) in children younger than 5 years, and currently no effective drugs are available for EV71. Thus, there is an urgent need to develop new drugs for the control of EV71 infection. In this study, LJ04 was extracted from Laminaria japonica using diethylaminoethyl cellulose-52 with 0.4 mol/l NaCl as the eluent, and its virucidal activity was evaluated based on its cytopathic effects on a microplate. LJ04 is composed of fucose, galactose, and mannose and mainly showed good virucidal activity against EV71. The antiviral mechanisms of LJ04 were the direct inactivation of the virus, the blockage of virus binding, disruptions to viral entry, and weak inhibitory activity against the nonstructural protein 3C. The two most important findings from this study were that LJ04 inhibited EV71 proliferation in HM1900 cells, which are a human microglia cell line, and that LJ04 can directly inactivate EV71 within 2 hr at 37°C. This study demonstrates for the first time the ability of a polysaccharide from L. japonica to inhibit viral and 3C activity; importantly, the inhibition of 3C might have a minor effect on the antiviral effect of LJ04. Consequently, our results identify LJ04 as a potential drug candidate for the control of severe EV71 infection in clinical settings. 相似文献
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Enterovirus 71 (EV71) is a major aetiological agent of hand, foot and mouth disease (HFMD). In recent years, several outbreaks in East Asia were associated with neurological complications and numerous deaths. An outbreak in Singapore in October 2000 afflicted thousands of children, resulting in four fatal cases from three of whom EV71 was isolated. The genomes of two representative EV71 strains isolated from a fatal case and a surviving patient were completely sequenced, and their nucleotide and amino acid sequences compared with known EV71 strains. The two outbreak strains were classified under genogroup B, together with those previously isolated in Singapore, Malaysia and Japan. Comparative sequence analysis of the two Singapore strains revealed 99% nucleotide similarity, while their deduced amino acid sequences were almost identical except for residue 1506 in the 3A non-structural region. Given that the outbreak involved closely related genetic variants of EV71, the broad spectrum of disease severity may be attributed to critical factors such as varying viral inoculation doses or differing host immune responses following infection, but is less likely to be due to the emergence of EV71 strains with heightened virulence. 相似文献
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Periodic outbreaks of hand, foot and mouth disease(HFMD) occur in children under 5 years old, and can cause death in some cases. The C4 strain of enterovirus 71(EV71) is the main pathogen that causes HFMD in China. Although no drugs against EV71 are available, some studies have shown that candidate vaccines or viral capsid proteins can produce anti-EV71 immunity. In this study, female BABL/c mice(6–8 weeks old) were immunized with virus-like particles(VLPs) of EV71 produced in yeast to screen for anti-EV71 antibodies. Two hybridomas that could produce neutralizing antibodies against EV71 were obtained. Both neutralizing m Abs(D4 and G12) were confirmed to bind the VP1 capsid protein of EV71, and could protect 95% cells from 100 TCID50 EV71 infection at 25 μg/m L solution(lowest concentration). Those two neutralizing m Abs identified in the study may be promising candidates in development for m Abs to treat EV71 infection, and utilized as suitable reagents for use in diagnostic tests and biological studies. 相似文献
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为探讨肠道病毒71型(EV71)VP4基因序列与手足口病(HFMD)不同临床类型之间的关系,分析重组蛋白EV71 VP4的抗原性,并初步探讨其与柯萨奇病毒A16(CA16)重组蛋白VP4是否存在交叉反应性,对2007~2009年从北京患HFMD儿童标本分离到的10株EV71的VP4基因进行克隆测序,运用生物学软件对测序结果进行比较分析,并选择其中1株与1株同期分离的CA16的VP4分别进行原核表达;用表达产物对189份正常体检的成人及来首都儿科研究所就医的非HFMD患儿血清中的IgG进行Western Blot检测,并分析14份确诊为EV71感染和12份CA16感染患者急性期血清中的IgM抗体。分析表明这10株EV71 VP4基因核苷酸同源性为94.20%~100.00%,所推导的氨基酸序列则完全相同,从重症与轻症患儿分离的毒株之间VP4的核苷酸序列未见一致性的差异,基于EV71 VP4基因核苷酸序列的进化树分析表明2007~2009年北京地区所流行的毒株均属于C4亚型;本研究中EV71和CA16的VP4核苷酸序列的同源性为69.60%,所推导的氨基酸序列的同源性为78.60%,在运用Western Blot检测189份血清中的VP4特异性IgG时,EV71 VP4的血清阳性率为38.10%,说明其具有良好的抗原性,CA16 VP4的血清阳性率为58.20%,两者差别具有显著统计学意义(2χ=15.30,P<0.01),提示EV71 VP4与CA16 VP4没有交叉反应性;在用表达的VP4检测已确诊为相应病毒的特异性IgM时,两者皆为阴性,提示感染后机体对VP1和VP4产生不同的反应。 相似文献
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为分析2009～2011年上海地区手足口病流行病学特征和病原构成,从国家疾病监测信息报告管理系统获取上海市2009～2011年手足口病流行病学资料;采用实时荧光反转录聚合酶链反应（RT-PCR）对来自上海18个区（县）的6676例手足口病病例标本进行肠道病毒核酸检测,对其中257份标本进行病毒分离;对27份人肠道病毒71型（HEV71）毒株进行VPl基因序列全长测定和分析。结果显示,2009～2011年上海市18个区（县）均有手足口病病例报道,地区分布元显著差异;≤5岁的婴幼儿为疾病高发年龄段;4～11月为发病高峰期。HEV71和柯萨奇病毒A组16型（CAl6）为主要病原,不同地区病原构成有所不同。实时荧光RT—PCR对6676例病例标本进行核酸检测,其中肠道病毒通用核酸检测阳性率为69．61％,HEV71和CAl6阳性率分别为38．83％和21．26％。对257份HEV71核酸阳性标本进行病毒分离,获得毒株57株,分离阳性率为22．18％。对其中27株进行VPl基因全长测序,均属C4a基因亚型。 相似文献
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目的:通过调查近年来我国肠道病毒EV-71型和柯萨奇病毒A16型流行株的全基因组序列,建立一种能够获得我国肠道病毒序列的通用扩增方法,为今后的手足口病流行病学分析、致病机理研究等打下基础。方法:收集我国近5年各地报道的肠道病毒流行株全基因组序列作为参考序列进行比对分析,在保守区设计通用引物,利用3'RACE、长距离PCR扩增及简并引物扩增肠道病毒全基因组序列,采用IonTorrentPGM二代测序仪对扩增产物进行深度测序,以对扩增方法进行验证和评价。结果:通过比对肠道病毒流行株序列设计了通用扩增引物,经二代测序实验获得了肠道病毒全基因组序列;以系列比例模拟混合病毒感染,该扩增方法能够同时获得2株肠道病毒的全基因组序列;能够完整地揭示肠道病毒重组情况。结论:建立了针对我国近年来肠道病毒流行株的通用全基因组扩增方法,在病毒培养液中肠道病毒的提取与扩增中显示了较高的灵敏度,能够反映混合病毒感染、重组病毒的情况。 相似文献
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Piyada Linsuwanon Jiratchaya Puenpa Sheng-Wen Huang Ya-Fang Wang John Mauleekoonphairoj Jen-Ren Wang Yong Poovorawan 《Journal of biomedical science》2014,21(1):16
Background
Human enterovirus 71 (EV71) is an important pathogen caused large outbreaks in Asian-Pacific region with severe neurological complications and may lead to death in young children. Understanding of the etiological spectrum and epidemic changes of enterovirus and population’s immunity against EV71 are crucial for the implementation of future therapeutic and prophylactic intervention.Results
A total of 1,182 patients who presented with the symptoms of hand foot and mouth disease (67.3%) or herpangina (HA) (16.7%) and admitted to the hospitals during 2008-2013 were tested for enterovirus using pan-enterovirus PCR targeting 5′-untranslated region and specific PCR for viral capsid protein 1 gene. Overall, 59.7% were pan-enterovirus positive comprising 9.1% EV71 and 31.2% coxsackievirus species A (CV-A) including 70.5% CV-A6, 27.6% CV-A16, 1.1% CV-A10, and 0.8% CV-A5. HFMD and HA occurred endemically during 2008-2011. The number of cases increased dramatically in June 2012 with the percentage of the recently emerged CV-A6 significantly rose to 28.4%. Co-circulation between different EV71 genotypes was observed during the outbreak. Total of 161 sera obtained from healthy individuals were tested for neutralizing antibodies (NAb) against EV71 subgenotype B5 (EV71-B5) using microneutralization assay. The seropositive rate of EV71-B5 was 65.8%. The age-adjusted seroprevalence for individuals was found to be lowest in children aged >6 months to 2 years (42.5%). The seropositive rate remained relatively low in preschool children aged > 2 years to 6 years (48.3%) and thereafter increased sharply to more than 80% in individuals aged > 6 years.Conclusions
This study describes longitudinal data reflecting changing patterns of enterovirus prevalence over 6 years and demonstrates high seroprevalences of EV71-B5 NAb among Thai individuals. The rate of EV71 seropositive increased with age but without gender-specific significant difference. We identified that relative lower EV71 seropositive rate in early 2012 may demonstrate widely presented of EV71-B5 in the population before account for a large outbreak scale epidemic occurred in 2012 with due to a relatively high susceptibility of the younger population. 相似文献11.
Shih‐Yeh Lin Chia‐Tsui Yeh Wan‐Hua Li Cheng‐Ping Yu Wen‐Chin Lin Jyh‐Yuan Yang Hsueh‐Ling Wu Yu‐Chen Hu 《Biotechnology and bioengineering》2015,112(10):2005-2015
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Ze Qin Lim Qing Yong Ng Yukei Oo Justin Jang Hann Chu Shi Yan Ng Siu Kwan Sze Sylvie Alonso 《EMBO reports》2021,22(6)
Enterovirus‐A71 (EV‐A71) has been associated with severe neurological forms of hand, foot, and mouth disease (HFMD). EV‐A71 infects motor neurons at neuromuscular junctions (NMJs) to invade the central nervous system (CNS). Here, we investigate the role of peripherin (PRPH) during EV‐A71 infection, a type III intermediate neurofilament involved in neurodegenerative conditions. In mice infected with EV‐A71, PRPH co‐localizes with viral particles in the muscles at NMJs and in the spinal cord. In motor neuron‐like and neuroblastoma cell lines, surface‐expressed PRPH facilitates viral entry, while intracellular PRPH influences viral genome replication through interactions with structural and non‐structural viral components. Importantly, PRPH does not play a role during infection with coxsackievirus A16, another causative agent of HFMD rarely associated with neurological complications, suggesting that EV‐A71 ability to exploit PRPH represents a unique attribute for successful CNS invasion. Finally, we show that EV‐A71 also exploits some of the many PRPH‐interacting partners. Of these, small GTP‐binding protein Rac1 represents a potential druggable host target to limit neuroinvasion of EV‐A71. 相似文献
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肠道病毒71型(enterovirus 71,EV71)为小RNA病毒科肠道病毒属成员,是引起手足口病的主要病原体之一。EV71流行广泛,其感染可引发中枢神经系统疾病,并造成重症手足口病,给公共卫生安全带来极大挑战。EV71的致病机制与病毒和宿主天然免疫系统的相互作用关系密切,涉及病毒逃逸干扰素反应、病毒抑制核因子κB(nuclear factorκB,NF-κB)信号通路及病毒与天然免疫细胞相互作用等多个环节。本文就近年来EV71与宿主天然免疫系统相互作用的研究进展进行综述。 相似文献
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The crystal structure of human cathepsin F and its implications for the development of novel immunomodulators 总被引：2，自引：0，他引：2
Cathepsin F is a lysosomal cysteine protease of the papain family, and likely plays a regulatory role in processing the invariant chain that is associated with the major histocompatibility complex (MHC) class II. Evidence suggests that inhibiting cathepsin F activity will block MHC class II processing in macrophages. Consequently, inhibitors of this enzyme may be useful in treating certain diseases that involve an inappropriate or excessive immune response. We have determined the 1.7A structure of the mature domain of human cathepsin F associated with an irreversible vinyl sulfone inhibitor. This structure provides a basis for understanding cathepsin F's substrate specificity, and suggests ways of identifying potent and selective inhibitors of this enzyme. 相似文献
18.
Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth.The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents.Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae.EV71 has been associated with an array of clinical diseases including hand foot and mouth disease (HFMD),aseptic meningitis,encephalitis and poliomyelitis-like acute flaccid paralysis.A large outbreak of HFMD due to highly neurovirulent EV71 emerged in Malaysia in 1997,and caused 41deaths amongst young children.In late 2000,a recurrence of an outbreak of HFMD occurred in Malaysia with S fatalities in peninsular Malaysia.Outbreak of HFMD due to EV71 recurred in 2003 with an unknown number of cases and mortalities.A similar outbreak of HFMD with 2 recorded deaths in young children occurred in peninsular Malaysia in late 2005 and this was followed by a larger outbreak in Sarawak (Malaysian Borneo) with 6 reported fatalities in the early part of 2006.The current on-going outbreak of HFMD started in peninsular Malaysia in epidemiological week 12 of 2010.As with other HFMD outbreaks in Malaysia,both EV71 and CA16 were the main aetiological viruses isolated.In similarity with the HFMD outbreak in 2005,the isolation of CA16 preceded the appearance of EV71.Based on the VP 1 gene nucleotide sequences,4 sub-genogroups of EV71 (C1,C2,B3 and B4) co-circulated and caused the outbreak of hand,foot and mouth disease in peninsular Malaysia in 1997.Two sub-genogroups (C1 and B4) were noted to cause the outbreak in 2000 in both peninsular Malaysia and Sarawak.EV71 of sub-genogroup B5 with smaller contribution from sub-genogroup C1 caused the outbreak in 2003.In the 2005 outbreak,besides the EV71 strains of sub-genogroup C1,EV71 strains belonging to sub-genogroup B5 were isolated but formed a cluster which was distinct from the EV71 strains from the sub-genogroup B5 isolated in 2003.The four EV71 strains isolated from clinical specimens of patients with hand,foot and mouth disease in the Sarawak outbreak in early 2006 also belonged to sub-genogroup B5.Phylogenetic analysis of the VP1 gene suggests that the EV71 strains causing the outbreak in Sarawak could have originated from peninsular Malaysia.Epidemiological and molecular data since 1997 show the recurrence of HFMD due to EV71 in Malaysia every 2 to 4 years.In each of the past outbreaks,more than one sub-genogroup of the virus co-circulate. 相似文献
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Hand foot and mouth disease is a febrile sickness complex characterized by cutaneous eruption (exanthem) on the palms and soles with simultaneous occurrence of muco-cutanous vesiculo-ulcerative lesions (enanthem) affecting the mouth. The illness is caused by a number of enteroviruses with coxsackievirus A16 and enterovirus 71 as the main causative agents. Human enterovirus 71 (EV71) belongs to the species Human enterovirus A under the genus Enterovirus within the family Picornaviridae. EV71 has been associa... 相似文献
20.
Rong-Rong Zhang Meng-Jiao He Chao Zhou Yan-Peng Xu Wei Tang Tian-Shu Cao Zheng-Jian Wang Mei Wu Tao Ming Yi-Jiao Huang Meng-Xu Sun Hui Zhao Yong-Qiang Deng Xiao-Feng Li Bin Wang Qing Ye Cheng-Feng Qin 《中国病毒学》2024,39(5):812-820
Human Enterovirus 71 (EV71) has emerged as one of the predominant causative agents of hand, foot and mouth disease (HFMD) with global impact. Despite the inactivated vaccine being licensed, other vaccine candidates based on advanced technology platforms are under development. In this report, we rationally designed and constructed two DNA-launched live attenuated vaccine candidates (pDL-EV71) under the control of specific promoters. In vitro and in vivo transfection with pDL-EV71 driven by the CMV promoter successfully yielded fully infectious EV71. More importantly, the administration of pDL-EV71 did not cause clinical symptoms following intracranial or intramuscular inoculation in neonatal and IFNα/βR-/- mice, demonstrating its safety profile. Moreover, a single-dose or two-dose immunization with pDL-EV71 elicited robust neutralizing antibodies against EV71 as well as an antigen-specific cellular response in mice. A single-dose immunization with 10 μg of pDL-EV71 conferred complete protection against lethal EV71 infection in neonates born to immunized maternal mice. Overall, our present results demonstrate that pDL-EV71 is a safe and effective vaccine candidate against EV71 for further development. 相似文献